Customising the plunge-freezing workflow for challenging conditions.

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Grid freezing is a critical step for successful cryo-transmission electron microscopy, and optimising freezing conditions is a considerable bottleneck in many projects. To improve reproducibility in grid preparation, temperature- and humidity-controlled chambers were built into the second generation of plunge-freezers, including the ThermoFisherScientific Vitrobot and Leica GP. Since then, for most published structures, the proteins were plunge-frozen from a cold, humid environment. This provides two benefits: many proteins are more stable at 4°C than room temperature, and both the low temperature and the humidity help control evaporation of the tiny drop of liquid. However, for optimal stability, certain samples may have different requirements. Here, we describe various (reversible) adaptations made to a Leica GP2 system to accommodate several samples with special handling requirements: a protein that is sensitive to both light and oxygen, a sample that needs to be kept at 37 °C throughout the plunge-freezing process, and a method to freeze a polymer that gels at 37 °C in its gelled state. While some of these methods are specific to these specimens, we hope sharing the ideas behind them will help people who are dealing with tricky protein samples.